This invention relates to an analyzer for the determination of the ABO unit and of the phenotype of blood.
More particularly, this invention relates to an analyzer of the type mentioned above, which is fully automatic and less expensive than the automatic analyzers available at the present time, though it is capable of performances similar to those of the latter.
As is well known, blood units are permanent characteristics of each individual and are determined by the genes that originate antigen and antibody systems.
An antigen represents a substance that, if introduced into an individual, stimulates an immune reaction characterized by the production of specific proteins: the antibodies. Thus, an antibody is a substance that reacts specifically with that antigen which has stimulated its production.
For the blood units, antigens are molecules which are immunologically reactive and are present on the surface of the red cells of each individual.
The blood unit antibodies are plasmatic proteins that can be classified as "natural antibodies", so that it is not possible to put into evidence a specific antigenic stimulus that has generated its product, and as "immune antibodies", which are synthesized by an organism in response to the introduction of a determined erythrocyte antigen.
The introduction of erythrocyte antigens with the subsequent production of immune antibodies and the consequent immune reaction can occur, in particular, during transfusion and pregnancy.
Accordingly, it is evident and well known to those who are skilled in the art that the determination of the antigen-antibody systems of blood unit is very important as regards the theory and practice of blood transfusion.
The most important blood units system, whose knowledge is fundamental for the efficiency and safety of blood transfusion, is the ABO system.
A further blood system of remarkable interest from an immunohematological standpoint is the Rhesus system (or Rh system), which collects together a wide series of different antigens.
Moreover, other minor antigen systems are present on the surface of the erythrocytes, said systems having less immune power than the blood unit systems mentioned above.
The contact between erythrocyte antigens and their corresponding antibodies can give rise to a reaction that is called agglutination and represents the most efficient and simplest way to make the antigen-antibody reaction visible.
Such agglutination reaction is exploited for the determination of blood units in normal laboratory practice.
In agglutination, two steps can be distinguished: in the first step the antibody molecule combines with the erythrocyte membrane antigens (the sensitization step), while in the second step the antibody forms bridge-links among different erythrocytes so determining the proper agglutination step.
At the present time, three methods are mainly employed for the determination of blood units. They are the manual method, the semi-automatic method (or the micromethod), and the automatic method.
The main manual procedures for determining blood units are:
the slide agglutination test: this is the test commonly adopted for the determination of the ABO and the Rh blood unit systems. It consists of a rapid test that is carried out with suspended red cells at 40-50% in plasma or an autologous serum, and is performed at room temperature if complete IgE type sera (reagents) are employed or if modified IgG type antibody-containing sera are employed, and at a temperature of about 37.degree. C. in the case of incomplete antibody sera which are made capable of giving agglutination by the hyperproteic medium;
the test-tube agglutination test: this is a test mainly employed for the determination of erythrocyte antigens with common incomplete sera. It is carried out with 5-10% suspended erythrocytes in a physiological solution, and the reading is performed after centrifuging the reagents.
Micromethods or semi-automatic methods make use of relatively concentrated erythrocytes suspensions containing very low amounts of the test-serum.
The procedure employed in micromethods is almost fully automatic.
In particular, a remarkable waste of time occurs with semiautomatic analyzers as the technician is to follow the various steps of the whole process, performing for instance the dispensing or pipetting operation and the transfer to the measuring operations.
The automatic method for determining the blood unit is carried out with very expensive analyzers, which as a consequence are inaccessible to the average or small laboratories.
Some analyzers of such type will be disclosed below, with particular reference to their technical features and performance.
GROUPAMATIC 2000 (KONTRON)
This is a 18-channel, automatic analyzer for the determination of blood units and of other immunohematological parameters, and is based on the photometric reading of agglutination.
The analyzing rate is 2,000 reactions/hour, with the possibility of analyzing 120 samples/hour over 18 channels, or 240 samples/hour over 9 channels.
It can be employed for:
A) The ABO determination, with discrimination of the weak variants of the antigen A (A3, Am, etc.);
B) The determination of Rh and of Du;
C) Screening of irregular antibodies by means of different techniques:
the saline medium PA1 the monomolecular medium PA1 bromelin-methylcellulose (BMC) PA1 trypsin-polybrene-citrate (TPC) PA1 low ionic strength hyperproteic medium (LIHP) PA1 1) the dilution station; PA1 2) the sampling and loading station; and PA1 3) the station for identifying the medium.
D) The determination of the anti A and anti B immune antibodies;
E) Screening of syphilis;
F) The determination of the Rh, Kell, Lewis, etc. phenotypes; and
G) The analysis of special antibodies (for instance, antitetanic antibodies).
Preparation of samples: Samples, collected together with EDTA into test-tubes, are centrifuged separately and put manually on the Groupamatic, in a driving system consisting of 120 containers assembled in series of 12:1 white and 11 black.
A sample tube and a dilution tube are associated with each container. A chain will take the samples through the following stations:
The dilution system: the dilution probe is inserted automatically into the sample tube. A pump sucks a volume of packed red cells, then it expels that volume together with a bromelin saline medium when the needle is positioned in front of the proper dilution tube.
The volumes of the red cells and of the physiological solution are adjustable by means of a computer program.
The washing operation between two samples is carried out externally by means of a washing ring and internally by the physiological solution employed for the dilution.
The sampling system: The samples go step by step towards the sampling station. There, the diluted red cells and the plasma are sucked through a double needle in the pipe and are taken to the proper cuvettes.
The reactants (antisera, special solutions, red cell-test) are stored in sets of a 9 beakers which are put on special racks.
The pipes carry the reagents which are removed from the beakers by the pump, into the proper cuvettes.
The recording of the identification number: The identification number of the samples is read by an optical system and transferred to the computer for storing the same.
The loading of samples: The immunohematological and serum reactions occur within the 216 cuvettes of the reaction disc.
There are 9 cuvettes and 24 sectors for each disc. The operator can make the decision of employing:
two sectors for each sample and 12 samples for each disc (up to 18 reactions for each sample); or
one sector for each sample and 24 samples for each disc (up to 9 reactions for each sample).
On the average, 10 reaction discs per hour are read.
The disc is loaded automatically by means of a "reagent" pump and a "sample" pump, said pumps distributing from 10 to 140.mu.liters per cuvette and from 1 to 5 reagents (optional).
An injection rotatable plate loads the first disc onto the first multitest station that causes in turn the loading of the second disc. Such a movable plate also allows the reagents to be loaded during the cycle.
The first stage consists in loading two sample sectors (diluted red cells and plasma) each time.
Two movable injectors deliver the selected volumes into each cuvette. The rapid loading of reagents occurs when the loading of the samples has been completed.
The processing occurring at the reaction disc: the multitest station generates at the disc:
a slow rotation (indexation);
a step-by-step motion for taking each cuvette sector below the injection plate and the photometers;
a fast rotation (centrifuging); and
a horizontal agitation.
The recording of reactions: at the end of each reaction cycle, the 9-cell photometer is positioned above the disc, where the reactions inside the cuvettes are read, one sector each time.
The photometric reading is based on the amount of light transmitted through the peripheral part and through the central part of the cuvette.
The results of such measurements are transmitted to the control unit and combined with the respective identification number.
The reactions are interpreted according to a pre-established logic that is shown on the display and is printed with its identification number.
The visual reading of the reactions: each sector on the disc corresponds to a specific reaction.
It is possible to observe the reactions both on the multitest station that can be lighted and on the disc itself after the same has been removed from the instrument.
The operator substitutes the disc with a clean disc and he starts a new cycle. The disc already employed can be rinsed or washed.
The photometric system: The control unit puts in correlation the contrast between the central and the peripheral light transmission measurements. The contrast is zero when the reaction is negative, and it increases when the reaction strength increases.
A set of thresholds is assigned to each channel, said thresholds discriminating the positive, the weakly positive and negative as well as the normal and abnormal reactions.
The abnormal reactions can be read visually by the operator and the result can be recorded on the file of the results by means of the video-terminal printer.
Such procedure keeps the number of samples to be tested again to a minimum.
The preparation of reactants: sera commercially available, once diluted in physiological solution or in macromolecular media, can be stored at 4.degree. C. for a week. The working schedule for determining the working dilution on a Groupamatic is as follows:
1) e.g.: anti-A titred 1/64 can be employed on said instrument when diluted to 4% concentration in saline medium or 2% in a macromolecular medium; and
2) anti-D titred 1/256 with bromelin-treated red cells is employed at 3% dilution in physiological solution.
TECHNICON AUTOGROUPER 16C
This is a 16-channel analyzer, which channels are programmed at the operator's choice for direct ABO and for indirect type determination; Rh type determination; phenotype determination; detection of irregular antibodies; detection of dangerous O donors; syphilis test. There is one channel for control functions.
It is possible to analyze 120 samples/hour and this analyzer allows complete computerization both of interpretation and of printing.
The identification of the sample occurs through the CODABAR system and laser scanning, and the possibility is provided of interfacing with a central computer.
The analyzer also allows flexible reagents and techniques to be employed on the basis of the selection of the reagents and of the agglutination temperature, i.e. 37.degree. C. or 18.degree. C.
This instrument is provided with dedicated automatic alarm systems and it shows a better monitor operation. Means are provided for signalling strong agglutinations.
The quantitative estimation of the single channel is also possible: if required, this is obtained through the measurement of the peak (height) of the channel selected.
The test-tubes containing the samples plus ACA or bar-marked EDTA are centrifuged and housed inside the chain "bandolier".
A probe double system sucks the upper portion (the plasma) and the lower portion (red cells).
Red cells are diluted automatically with physiological solution and are treated with enzymes in order to increase their sensitivity.
The main channel of the red cells is divided into sub-channels for determining ABO types and for direct determination of Rh.
The proper antisera are formed inside the channel together with PVP or methylcellulose in order to increase the formation of the agglutinate (rouleaux).
The suspension of samples is kept by air segmentation. The agglutinated materials formed within the reaction loops are dispersed by the successive addition of physiological solution and by a short resolution time.
The various agglutinates that are deposited into a button are sucked off (decanted) in two different moments.
The flow is then freed from air bubbles and the optical density of the remainder liquid is determined continuously by means of an optical device.
The plasma channel is in turn divided for the indirect type determination against red cells and for the determination of antibodies, the detection of dangerous O's and the ART test of syphilis.
Moreover, the "Filter Paper Backup" is provided, which is an optional system for the visual identification and backup of the ODU (Optical Detection Units) system in the case of electronic faults.
At the first decanting operation, the agglutinated materials are deposited onto filter paper instead of conveying them to the waste discharge point.
The selection of temperatures: the channels are kept at constant temperatures of 37.degree. C. or 18.degree. C. according to the operator's frequency.
The red cell suspension and the antisera are mixed and kept at a constant temperature of 12.degree. C.
The very brain of the unit consists of an INTEL microprocessor that is so programmed as to also receive signals from the 16 optical reading systems to decide about the ABO and Rh grouping and about the occurrence or not of an agglutination in each channel.
After making such decisions, the microprocessor correlates the results so obtained with the sample number which is read by a laser source on a bar code and transmitted from an interface to the same microprocessor.
The information is passed to the printer that gives a list of each sample according to the identification bar code and to its sequential number.
Similar signals for each channel are passed from the microprocessor to the recorder for their quantification.
The agglutination strength depends on the peak height. The positive signals are also transmitted to the event marker that acts as a monitor and points out the channel phase and as a second signal check.
Moreover, the microprocessor selects the sample washing time of the sampler, selects the discrimination levels of the optical units, functions as a monitor of the instrument performance with the carryover and phase check, and it can be interfaced with any other computer and can be programmed within quite wide limits according to the single uses.
OLYMPUS PK 7100
This automatic analyzer carried out the analyses of 120 samples per hour, and the first sample goes out after one incubation hour at 37.degree. C. It comprises microplates bearing 10 samples and 16 channels. Antisera after suitable dilution and the red cells in suspension are drawn and dispensed automatically by means of 16 syringes.
The twelve cuvettes containing the antisera and the red cells are stirred automatically by a comb-like system.
The sampling system is made up of two needles, the first one of which sucks the plasma while the other one sucks the red cells. The latter are then diluted automatically inside a well, whereas the plasma is diluted inside three different wells.
The analyzer is provided with various programs for defining the analysis profile to be exploited in the determination of the red cell type.
Once the automatic distribution of antisera and/or red cells as well as of the samples to be analyzed has occurred, the microplate is automatically taken into a chamber kept at a constant temperature of 37.degree. C. where the microplate will be kept for an hour.
The microplate is then transferred automatically into the reading seat.
The reading is carried out by means of a photocell (for each channel) that reads the difference in brightness between the central and the peripheral zone of the well (like GROUPMATIC 2000-KONTRON).
Moreover, there is an illuminated plane that allows the visual check of the results given by the machine. The plates can be employed again after accurate washing. There is no graphic detection for checking the existence of the agglutination reaction.
The final report gives just the identification number of the sample and the result obtained. Thus the analyzer in question is a very cumbersome apparatus that is sold at a very high price and that cannot carry out the Coembs test.
As a matter of practice, the automatic analyzers disclosed so far, that are capable of decidedly high technical performances, can be only employed in very large sanitary structures, in particular because of their high production costs that derive mainly from the complex realization of the analyzer which is to allow all analysis required to be performed.
Indeed, this is quite a severe problem, also because of the delicate character of such type of analysis and because of the consequences, which are also of penal kind, of an erroneous result.
Accordingly, it is quite evident that it is important to have an analyzer which, by quality and reliability, is capable of giving performances similar to those of the automatic analyzers commercially available at present, but which can be realized at a cost 3-5 times lower, so that such analyzers will be accessible even to small analytical laboratories.
The Applicant, in order to reach such a technical result, has investigated and realized a fully automatic analyzer, whose "philosophy" of reading of the results, which is completely new in the specific field, though it has been adopted in other fields, has allowed an analyzer to be technically achieved that, though lacking some technical performances of the best automatic analyzers, is structurally simpler and can be manufactured at remarkably reduced costs.
The reading of the results in the semi-automatic and automatic analyzers presently available is realized by determining the number of red cells that have become agglutinated and deposited onto the bottom of the test tubes. This type of reading requires a double determination on the bottom of the test-tube, with a vertical determination (the light coming from the bottom and the reading device being at the top) and a horizontal determination to obtain a complete result.